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The invention is directed to the field of transgenic mice containing a disrupted PTP-1B gene. The mice may contain a disruption in either one or both copies of the PTP-1B gene. In the case of mice containing a disruption in both copies of the PTP-1B gene, such mice lack detectable expression of PTP-1B protein.
Protein tyrosine phosphatases (PTPases) are a large family of transmembrane or intracellular enzymes that dephosphorylate substrates involved in a variety of regulatory processes (Fischer et al., 1991, Science 253:401-406). Protein tyrosine phosphatase-1B (PTP-1B) is a xcx9c50 kd intracellular protein present in abundant amounts in various human tissues (Charbonneau et al., 1989, Proc. Natl. Acad. Sci. USA 86:5252-5256; Goldstein, 1993, Receptor 3:1-15). Like other PTPases, PTP-1B has a catalytic domain characterized by the sequence motif (I/V)HCXAGXXR(S/T)G (SEQ.ID.NO.:1), containing arginine and cysteine residues that are critical to the enzyme""s activity (Streuli et al., 1990, EMBO J. 9:2399-2407; Guan et al., 1990, Proc. Natl. Acad. Sci. USA 87:1501-1505; Guan and Dixon, 1991, J. Biol. Chem. 266:17026-17030). The amino terminal 35 amino acid residues of PTP-1B localize the protein to the endoplasmic reticulum (Frangioni et al., 1992, Cell 68:545-560).
Determining which proteins are substrates of PTP-1B has been of considerable interest. One substrate which has aroused special interest is the insulin receptor. The binding of insulin to the insulin receptor results in autophosphorylation of the receptor, most notably on tyrosines 1146, 1150, and 1151 in the kinase catalytic domain (White and Kahn, 1994, J. Biol. Chem. 269:1-4). This causes activation of the insulin receptor tyrosine kinase, which phosphorylates the various insulin receptor substrate (IRS) proteins that propagate the insulin signaling event further downstream to mediate insulin""s various biological effects.
Seely et al., 1996, Diabetes 45:1379-1385 (Seely) studied the relationship of PTP-1B and the insulin receptor in vitro. Seely constructed a glutathione S-transferase (GST) fusion protein of PTP-1B that had a point mutation in the PTP-1B catalytic domain. Although catalytically inactive, this fusion protein was able to bind to the insulin receptor, as demonstrated by its ability to precipitate the insulin receptor from purified receptor preparations and from whole cell lysates derived from cells expressing the insulin receptor.
Ahmad et al., 1995, J. Biol. Chem. 270:20503-20508 used osmotic loading to introduce PTP-1B neutralizing antibodies into rat KRC-7 hepatoma cells. The presence of the antibody in the cells resulted in an increase of 42% and 38%, respectively, in insulin stimulated DNA synthesis and phosphatidyinositol 3xe2x80x2 kinase activity. Insulin receptor autophosphorylation and insulin receptor substrate-1 tyrosine phosphorylation were increased 2.2 and 2.0-fold, respectively, in the antibody-loaded cells. The antibody-loaded cells also showed a 57% increase in insulin stimulated insulin receptor kinase activity toward exogenous peptide substrates.
Until the present invention, studies of the interaction of PTP-1B and the insulin receptor were limited to studies conducted on cell-free preparations of PTP-1B or in cultured cell lines. Therefore, such studies did not address the issue of whether PTP-1B activity affects the regulation of the insulin receptor in a way that results in physiological effects on glucose metabolism, triglyceride metabolism, or weight gain in living mammals. Because of the complexity of the regulation of the insulin receptor and its interactions with proteins such as PTP-1B, there is a need to study this regulation in an environment that is as close as possible to that of a living mammal. The knockout mice of the present invention are useful in helping to meet this need. The knockout mice of the present invention also are useful in that they provide an animal model that can be used in the design and assessment, in a living mammal, of compounds that modulate insulin receptor activity.
The present invention provides mice that have had their PTP-1B genes disrupted by targeted homologous recombination. When both copies of their PTP-1B genes are disrupted, the mice have no detectable PTP-1B protein, yet appear to be physiologically normal. However, in the fed state, the mice have slightly lower glucose levels and half the level of circulating insulin as their wild-type littermates. In glucose and insulin tolerance tests, the mice show increased insulin sensitivity. When fed a high fat, high carbohydrate diet, the mice, although much more insulin-sensitive than wild-type controls, are obesity-resistant.
Methods of making the mice and cell lines derived from the mice are also provided.
The present invention also provides methods of identifying inhibitors of the enzymatic activity of PTP-1B as well as inhibitors identified by such methods.